5-(beta-D-Ribofuranosyl)isocytosine (psi l Cyd), a C-nucleoside, has been shown to be active against P815 leukemia in mice. In P815 cells treated with [2-14C]psi l Cyd, we have detected radioactivity in nucleotide fractions and in RNA and DNA. Degradation to nucleosides of the labeled triphosphate nucleotide fraction and of RNA showed that the radioactivity present was chromatographically identical to psi l Cyd. Half-saturation concentrations for the incorporation of [2-14C]psi l Cyd into the triphosphate nucleotide fraction and into RNA and DNA were 370, 280, and 94 microgram/ml, respectively, which were greater than 100-fold higher than those for tritiated cytidine. The incorporation of psi l Cyd was competitively inhibited by cytidine. Phosphorylation and incorporation of psi l Cyd into nucleic acids of P815 cells and of a P815 subline resistant to 1-beta-D-arabinofuranosylcytosine are about 2- to 20-fold higher than in P815 sublines resistant to psi l Cyd or to both 5-azacytidine and 1-beta-D-arabinofuranosylcytosine. These data suggest that the phosphorylation of psi l Cyd and possibly its incorporation into nucleic acids are essential for therapeutic activity in P815 leukemias. In vitro metabolic studies also suggest that psi l Cyd and 5-azacytidine are cross-resistant and that P815 cells resistant to psi l Cyd are collaterally sensitive to 1-beta-D-arabinofuranosylcytosine. These predictions were confirmed by therapeutic experiments carried out in mice bearing P815 leukemias.